Target antigen discovery, phenotypic screens and use thereof for identification of target cell specific target epitopes

ABSTRACT

The invention provides methods and compositions for identifying binding polypeptides (e.g., antibodies or antigen binding fragments thereof) that specifically binds to a cell-surface antigen. The methods of the invention generally comprise contacting a variegated nucleic acid-display library of binding polypeptides with a cell-surface antigen displayed on the exterior surface of a cell; and isolating from the library at least one library member that specifically binds to the cell-surface antigen on the exterior surface of the cell.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/448,567, filed Jun. 21, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/897,892, filed Dec. 11, 2015, now U.S. Pat. No.10,370,651, which is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/US2014/043454, filed Jun. 20, 2014, which claimspriority to U.S. Provisional Patent Application No. 61/840,583, filedJun. 28, 2013, the entire contents of which are incorporated herein intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 29, 2022, isnamed 732235_XBI-010USCON2_ST26.xml and is 13,691 bytes in size.

BACKGROUND

Binding polypeptides, such as antibodies and fragments thereof, arecommercially important as therapeutic and diagnostic agents. Traditionalmethods of screening for binding polypeptide generally employ solubleantigens. However, for certain cell-surface antigens, conformationalepitopes on these antigens are altered when the antigens are solubilizedfrom the plasma membrane, resulting in a failure to generate bindingpolypeptides that can recognize the native antigen. Accordingly, thereis a need in the art for novel methods of screening for bindingpolypeptides that can specifically bind to cell-surface antigens intheir native conformation.

SUMMARY

The invention provides methods and compositions for identifying bindingpolypeptides (e.g., antibodies or antigen binding fragments thereof)that specifically binds to a cell-surface antigen. The methods of theinvention generally comprise contacting a variegated nucleicacid-display library of binding polypeptides with a cell-surface antigendisplayed on the exterior surface of a cell; and isolating from thelibrary at least one library member that specifically binds to thecell-surface antigen on the exterior surface of the cell. The methodsand compositions of the invention are particularly advantageous in thatthey allow for the rapid identification of binding polypeptides thatbind to native forms of the target cell surface antigen. These methodsand compositions also allow for identification of novel, therapeuticallyuseful cell-type specific antigens or epitopes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of exemplary DNA display compositions andscreening methods of the invention.

FIG. 2 is a schematic of exemplary DNA display compositions andscreening methods of the invention.

FIG. 3 is a schematic of exemplary target cell screening strategiesemployed in the methods of the invention.

FIG. 4 is a schematic of exemplary parallel screening and deepsequencing strategies employed in the methods of the invention.

FIG. 5 is a schematic of exemplary parallel screening and deepsequencing strategies employed in the methods of the invention.

FIG. 6 is a schematic of exemplary parallel screening and deepsequencing strategies employed in the methods of the invention.

FIG. 7 is a schematic of the results of exemplary parallel screeningstrategies employed in the methods of the invention.

FIG. 8 depicts a graph showing the results of a FACS based binding assayof high affinity VH molecules selected using the methods of theinvention.

FIG. 9 depicts graphs showing the differential binding of VH moleculesselected using the methods of the invention.

FIG. 10 depicts graphs showing the differential binding of VH moleculesselected using the methods of the invention.

DETAILED DESCRIPTION I. Definitions

As used herein, the term “nucleic acid display library” refers to anyart recognized in vitro cell-free phenotype-genotype linked display,including, without limitation those set forth in, for example, U.S. Pat.Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 6,518,018; 7,125,669;6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804;6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926;6,416,950; 6,660,473; 6,312,927; 5,922,545; and 6,348,315, and inWO2010/011944, which are all hereby incorporated by reference in theirentirety.

As used herein, the term “antigen” refers to the molecule recognized bya binding polypeptide.

As used herein, the term “specifically binds to” refers to the abilityof a binding molecule (e.g., a VH or VL domain) to bind to an antigenwith an affinity of at least about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, or more, and/or bind to a targetwith an affinity that is at least two-fold greater than its affinity fora nonspecific antigen.

As used herein, the term “antibody” refers to immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated VH) and a heavy chain constant region. The heavychain constant region comprises three domains, CH1, CH2 and CH3. Eachlight chain comprises a light chain variable region (abbreviated VL) anda light chain constant region. The light chain constant region comprisesone domain (CL1). The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR).

As used herein, the term “antigen-binding portion” of an antibodyincludes any naturally occurring, enzymatically obtainable, synthetic,or genetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and optionally constantdomains. Non-limiting examples of antigen-binding portions include: (i)Fab fragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding portion.”

As used herein, the terms “VH domain” and “VL domain” refer to singleantibody variable heavy and light domains, respectively, comprising FR(Framework Regions) 1, 2, 3 and 4 and CDR (Complementary DeterminantRegions) 1, 2 and 3 (see Kabat et al. (1991) Sequences of Proteins ofImmunological Interest. (NIH Publication No. 91-3242, Bethesda).

II. Cell Surface Antigens

In certain aspects, the invention provides methods of identifying abinding polypeptide that specifically binds to a cell-surface antigen.

Any antigen that is capable of being displayed on the surface of a cellcan be employed in the methods of the invention, including withoutlimitation, protein, glycan, and/or lipid antigens. In certainembodiments, the antigen is a naturally occurring molecule. Suitable,non-limiting examples of naturally occurring antigens includetransmembrane proteins (e.g., G-protein coupled receptors) andGPI-anchored proteins. In certain embodiments, the antigen is a non-naturally occurring recombinant or synthetic antigen. Suitable,non-limiting examples of naturally occurring antigens include chimericantigens comprising portions from different antigen molecules. Incertain embodiments, the identity of the antigen is known prior topreforming the methods of the invention. In certain embodiments, theidentity of the antigen is unknown prior to preforming the methods ofthe invention.

The cell surface antigens employed in the methods of the invention canbe displayed on any cell or cell-like particle (e.g., lipid vesicle). Incertain embodiments, the cell is a cell type that naturally expressesthe cell-surface antigen. In certain embodiments, the cell is arecombinant cell that is engineered to heterologously express thecell-surface antigen. In certain embodiments, the cell is adisease-associated variant of a normal cell (e,g, a tumor cell).

III. Binding Polypeptides

In certain aspects, the invention provides methods of identifying abinding polypeptide that specifically binds to a cell-surface antigen.

Any type of binding polypeptide can be employed in the methods of theinvention including, without limitation antibodies, or fragmentsthereof, and immunoglobulin-like domains. Suitable immunoglobulin-likedomains include, without limitation, fibronectin domains (see, forexample, Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which isincorporated by reference herein in its entirety), DARPin (see, forexample, Stumpp et al. (2008) Drug Discov. Today 13 (15-16): 695-701,which is incorporated by reference herein in its entirety), Z domains ofprotein A (see, Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which isincorporated by reference herein in its entirety), Lipocalins (see, forexample, Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which isincorporated by reference herein in its entirety), Affilins (see, forexample, Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-85, which isincorporated by reference herein in its entirety), Affitins (see, forexample, Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058-68,which is incorporated by reference herein in its entirety), Avimers(see, for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-61, which is incorporated by reference herein in its entirety),Fynomers, (see, for example, Grabulovski et al. (2007) J. Biol Chem 282(5): 3196-3204, which is incorporated by reference herein in itsentirety), and Kunitz domain peptides (see, for example, Nixon et al.(2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which is incorporatedby reference herein in its entirety). In certain embodiments, thebinding polypeptide is antibody VH or VL domain.

IV. Cell Surface Display Methods

In certain aspects, the invention provides a method of identifying abinding polypeptide that specifically binds to a cell-surface antigen,the method comprising: (a) contacting a variegated nucleic acid-displaylibrary of binding polypeptides with a cell-surface antigen displayed onthe exterior surface of a first cell type; and (b) isolating from thelibrary at least one library member that specifically binds to thecell-surface antigen on the exterior surface of the first cell type,thereby identifying a binding polypeptide that specifically binds to thecell surface antigen. In certain embodiments, prior to step (a), thevariegated nucleic acid-display library of binding polypeptides iscontacted with a second cell type that does not display the antigendisplayed on the exterior surface, in order to pre-clear the library ofbinding polypeptides that do not specifically bind to the antigen.

In certain aspects, the invention provides a method of identifying abinding polypeptide that specifically binds to a cell-surface antigen,the method comprising: (a) contacting a variegated nucleic acid-displaylibrary of binding polypeptides with a first cell type expressing acell-surface antigen, and isolating from the library at least onelibrary member that specifically binds to the first cell type; (b)contacting the variegated nucleic acid-display library of bindingpolypeptides with a second cell type that does not express the cellsurface antigen, and isolating from the library at least one librarymember that specifically binds to the second cell type; and (c)selecting library members that specifically bind to the first cell typebut not to the second cell type, thereby identifying a bindingpolypeptide that specifically binds to the cell surface antigen.

Suitable nucleic acid-display libraries for use in the methods of theinvention are set forth in, for example, U.S. Pat. Nos. 7,195,880;6,951,725; 7,078,197; 7,022,479; 6,518,018; 7,125,669; 6,846,655;6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804; 6,429,300;6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926; 6,416,950;6,660,473; 6,312,927; 5,922,545; and 6,348,315, and in WO2010/011944,which are all hereby incorporated by reference in their entirety. Incertain embodiments, the variegated nucleic acid-display library is aDNA display library. In one embodiment, the nucleic acid-display libraryis a DNA display library described herein or in WO2010/011944, which ishereby incorporated by reference in its entirety.

In certain embodiments, each member of the DNA-display library comprisesa binding polypeptide linked through an intervening DNA linker to a DNAcoding sequence encoding the binding polypeptide, wherein the DNA linkercomprising a restriction endonuclease site (see e.g., FIG. 1 ). Anyrestriction endonuclease site can be employed. In one particularembodiment, the restriction endonuclease site is not present in the DNAcoding sequence of members of the DNA-display library, thus avoidingcleavage of the DNA coding sequence upon restriction endonucleasedigestion of the library members. In one particular embodiment, therestriction endonuclease site is a Not1 site.

In certain embodiments, it is desirable to physically separate the DNAcoding sequence of the isolated library members from the linked bindingpolypeptide. Any methods of physical separation can be employed. Wherethe isolated library members comprise a DNA linker comprising arestriction endonuclease site (see e.g., FIG. 1 ), the physicalseparation can be achieved by restriction endonuclease digestion of theisolated library members. The resultant liberated DNA coding sequencescan be further separated from the cell/binding polypeptide complexes byany art recognized method, e.g., centrifugation .

In certain embodiments, it is desirable to physically separate theintact isolated library members from the from the first and/or secondcell type. Any methods of physical separation can be employed. Incertain embodiments, the isolated library members are separated from thefirst or second cell type by enzymatic cleavage of the cell-surfaceantigen. Any methods of enzymatic cleavage of the antigen can beemployed, e.g., protease, lipid, and/or glycosidase enzymatic cleavage.In certain embodiments, where the cell-surface antigen is attached tothe cell surface by a glycolipid anchor, the isolated library membersare separated from the first or second cell type by phospholipasecleavage of the glycolipid anchor. The resultant liberated isolatedlibrary members can be further separated from the first or second celltype by any art recognized method, e.g., centrifugation.

Once the library members that specifically bind to the first and/orsecond cell type have been isolated, the DNA coding sequence of thesemolecules can be determined. Accordingly, in certain embodiments, themethods of the invention further comprise the step of determining theDNA coding sequence of at least a portion of the isolated librarymembers. Any art recognized means for DNA sequence determination can beemployed. In one particular embodiment, the DNA coding sequence isdetermined by single molecule, deep sequencing techniques (e.g.,pyrosequencing). Single molecule, deep sequencing techniques are wellknown in the art (see e.g., those described in U.S. Pat. No. 6,210,891,which is hereby incorporated by reference in its entirety). In certainembodiments, where the binding polypeptides are antibodies, or antigenbinding fragments thereof, the DNA coding sequence of the CDR3 region isdetermined. In certain embodiments, the DNA coding sequences of thelibrary member that bind to the first and second cell types aredetermined. Library members that specifically bind to the first celltype but not to the second cell type are considered to comprise bindingpolypeptides that specifically bind to an antigen specific for the firstcell type.

Once a binding polypeptide that specifically binds to the cell-surfaceantigen has been identified, it can be heterologously expressed in vitro(e.g., in cells or in a cell-free expression system) or in vivo (e.g.,in a transgenic animal). Accordingly, in certain embodiments, themethods of the invention further comprise the step of heterologouslyexpressing in vitro (e.g., in cells or in a cell-free expression system)or in vivo (e.g., in a transgenic animal), the identified bindingpolypeptide.

In certain embodiments, the identity of the antigen is known prior topreforming the methods of the invention. However, it is not necessary toknow the identity of the antigen. Indeed, in certain embodiments, theidentity of the antigen is unknown prior to preforming the methods ofthe invention. Thus, in this latter case, the methods of the inventionallow for the identification of novel antigens and epitopes present onthe surface of a cell type of interest (e.g., a tumor cell).

In certain embodiments, the methods disclosed herein comprise theselection of binding polypeptides that are capable of functionalinternalization upon binding to the cell surface antigen. Such bindingpolypeptides are particularly useful in the production of drugconjugates because they allow for the delivery of a cytotoxic drug tothe interior of a target cell. Any methodology for screening forfunctional internalization can be employed. For example, the variegatednucleic acid-display library of binding polypeptides can be contactedwith target cells under conditions that to allow for binding polypeptideinternalization (e.g., for about for 1-2 hours at 37° C.). The cells canthen washed and lysed with cell lysis buffer in the presence of proteaseinhibitors. The internalized library members can then be ethanolprecipitated and the DNA coding sequences enriched by PCR amplification.

The methods disclosed herein can be applied to any target epitopediscovery process. For example, target epitopes can include: homingdomains for inflammation; tumor specific target epitopes from primarytumors with or without resistance to treatment, tumor cell lines, andtumors that harbor any mutations that may result in neoepitopes; andother disease specific epitopes that mediate disease specificmalfunction and be targeted for biologic therapy

The methods disclosed herein can also be applied for biomarker discoveryto monitor the presence or absence of particular cell surface epitopesover a course of a drug treatment to patients. The antibodies derivedfrom the biomarker discovery can also be used as tools for biomarkerdetection.

Additionally or alternatively, the methods disclosed herein can beapplied to target or epitope discovery in other species, such as intransgenic animals and animal disease models.

V. Exemplification A. Summary

A fully human antibody VH library obtained from bone marrow, peripheralblood and splenocytes of human donors was constructed and numerous highaffinity and specific VH binders to multiple targets were identifiedusing dsDNA display technology. Target cell specific epitopes wereidentified by live cell selection and deep sequencing analysis usinghuman VH library and dsDNA display technology. Strategies of parallel,differential selections and target cell selective selections wereapplied in these methods (see FIGS. 4, 5, 6 ). The tabulation of theCDR3 frequency from deep sequencing of all pools across all roundspredicted the selectivity of the VH clones. High affinity VHs wereidentified that selectively bind to target cells and related cell types.

B. Library Engineering and Selection Methods for Live Cell EpitopeDiscovery

Two methods were developed to effectively recover library members thatbound to live cells.

The first method involved stripping off binders from live cells byrestriction digestion of the DNA fused to bound antibodies. This methodenables the full recovery of the VHs bound to all epitopes on cells. AC-terminal of VH DNA library was engineered to carry a NotI restrictionsite (see FIG. 1 ). There are no NotI sites in naive VH frameworks andtherefore only full length VH binders are eluted from cells forsubsequent amplification. The NotI restriction digestion buffer wastested on live cells, and with up to a 2 hour incubation at 37C thecells were viable. The efficiency of the NoI digestion was high.Following the binding of the library to cells for 1 hour at 4C, thecells were washed and digested with NotI buffer at 37C for 1 hour, thecells were then spun down, supernatant (containing bound VH DNA) wascollected for PCR amplification (see FIG. 1 ).

The second method is to clip off VH binders from live cells usingphospholipase C (PLC) digestion. This method enables the elution of theVHs that bound to the epitopes of any GPI anchored membrane protein(i.e., a subset of epitopes). The PLC clipping efficiency is high, asvalidated on FACS with control molecule. After incubation of librarywith cells for 1 hour at 4C, the cells were washed and incubated withPLC at 37C for 15 mins. The cells were subsequently spun down and thesupernatant, containing fusion VH complexed with extracellular domain ofthe GPI anchored membrane protein, was PCR amplified (see FIG. 2 ).

C. Parallel Selections, Differential Selections and Target CellSelective Selections on Target and Related/Undesired Cell Types

Master naive fusion library was produced according to the protocol setforth in WO2010/011944 (which is hereby incorporated by reference in itsentirety). For first round of selection, the purified fusion library wassplit equally into multiple libraries that carry the same diversity forall selection branches (see FIG. 5 ).

Primary cells, obtained from normal donors or patients, were eitherthawed fresh or isolated from cell culture flasks, following standardcell biology protocols. The cells were then recovered for 1 hour in fullmedia at 37C followed by blocking in selection buffer for 30 mins onice. All the selections were carried out on ice to prevent antibody andtarget internalization.

For parallel selections, libraries were pre-cleared with 200 ul ofpre-blocked streptavidin beads and 200 ul of pre-blocked hIgG-Epoxybeads for 30 mins at room temperature sequencially to remove any misfoldand sticky library members. The pre-cleared libraries were then chilledon ice and subjected to pre-blocked cells and incubated on ice for 1hour.

For target cell selective selections, pre-clearance was performed onundesired and closely related cell types for 1 hour on ice to remove anynon-selective binders and then subjected on target cells.

At selection round 4, differential selection methods were applied to thebranches of selection on target cells (with and without pre-clearance oncells). In this round, libraries were split into multiple tubes andbound to each cell type and patient's cells from different stage of thediseases in parallel. This strategy allowed for direct comparison oftarget cells versus other cell types by deep sequencing analysis andidentification of binders recognizing different epitopes that arose withdisease progression (see FIG. 6 ).

For all selection branches, after binding, cells were washed with 10 mLof binding buffer and subject to either NotI restriction digestion torecover all binders to membrane protein or PLC clipping to recoverbinders to GPI anchored membrane proteins as described above.

D. Deep Sequencing Analysis to Predict Selective Binders to Target Cells

After each round of selection, binding pools were PCR amplified. TheHCDR3 of each binding pool was lifted up by PCR with oligos primingC-terminal of framework 3 and N-terminal of framework 4 of VH fragments.These HCDR3 fragments derived from individual selection rounds andbranches were then tagged with specific DNA bar code used for Illuminasequencing by PCR. The tagged HCDR3 were pooled and sent for highthroughput sequencing with Hi Seq technology. The round 4 binding poolsfrom target cells were also tagged with DNA bar code and submitted for454 sequencing to get full length VH sequences.

The sequences were deconvoluted based on the DNA bar code aftersequencing. Millions of sequences derived from each selection round andselection branch were tabulated by comparing the frequency of aparticular CDR3 sequence present at different rounds and selectionbranches. The criteria used for identification of selective binderswere: 1) specific enrichment of a CDR3 sequence from earlier round tolater round on target cells, not on control or close related cell types;2) higher frequency on target specific cell type and low on control orclosely related cell type at differential selection round (see FIGS. 7); and 3) sequences not present in other target or cell selections fromother programs in database. The selective clones identified by Illuminasequencing were then synthesized based on the 454 full length sequenceinformation.

E. Production, Purification and FACS Binding Assays

The binding pools and synthesized VHs were then subcloned into pET22bexpression vectors. The VHs were produced in BL-21 E. coli cells andpurified through C-terminal His tag using standard protocols. FACS assaywas performed to assess the binding and selectivity of VHs to differentcell types and the EC50 of the binders. High affinity and selective VHbinders were identified through the live cell selection process (seeFIGS. 8, 9 and 10 ).

1. A method of identifying a binding polypeptide that specifically bindsto a cell-surface antigen, the method comprising: a. contacting avariegated nucleic acid-display library of binding polypeptides with acell-surface antigen displayed on thcan exterior surface of a first celltype; and b. isolating from the library at least one library member thatspecifically binds to the cell-surface antigen on the exterior surfaceof the first cell type, thereby identifying a binding polypeptide thatspecifically binds to the cell surface antigen.
 2. The method of claim1, wherein prior to step (a), the variegated nucleic acid-displaylibrary of binding polypeptides is contacted with a second cell typethat does not display the antigen displayed on the exterior surface. 3.A method of identifying a binding polypeptide that specifically binds toa cell-surface antigen, the method comprising: a. contacting avariegated nucleic acid-display library of binding polypeptides with afirst cell type expressing a cell-surface antigen, and isolating fromthe library at least one library member that specifically binds to thefirst cell type; b. contacting the variegated nucleic acid-displaylibrary of binding polypeptides with a second cell type that does notexpress the cell surface antigen, and isolating from the library atleast one library member that specifically binds to the second celltype; and c. selecting library members that specifically bind to thefirst cell type but not to the second cell type, thereby identifying abinding polypeptide that specifically binds to the cell surface antigen.4. The method of claim 1, wherein the variegated nucleic acid-displaylibrary is a DNA display library.
 5. The method of claim 4, wherein eachmember of the DNA-display library comprises a binding polypeptide linkedthrough an intervening DNA linker to a DNA coding sequence encoding thebinding polypeptide, wherein the DNA linker comprising a restrictionendonuclease site.
 6. The method of claim 5, wherein the restrictionendonuclease site is not present in the coding sequence of members ofthe DNA-display library.
 7. The method of claim 2, wherein the methodfurther comprises physically separating the DNA coding sequence and thelinked binding polypeptide of the isolated library members.
 8. Themethod of claim 2, wherein the method further comprises separating theintact isolated library members from the first or second cell type. 9.The method of claim 8, wherein the isolated library members areseparated from the first or second cell type by enzymatic cleavage ofthe cell-surface antigen.
 10. The method of claim 9, wherein thecell-surface antigen is attached to the cell surface by a glycolipidanchor and the isolated library members are separated from the first orsecond cell type by phospholipase cleavage of the glycolipid anchor. 11.The method of claim 1, wherein the method further comprises determiningthe DNA coding sequence of at least a portion of the isolated librarymembers.
 12. The method of claim 11, wherein the DNA coding sequence isdetermined by pyrosequencing.
 13. The method of claim 1, where theantigen is a naturally occurring protein, glycan or lipid.
 14. Themethod of claim 1, where the antigen is a glycophosphatidylinositol(GPI) anchored protein.
 15. The method of claim 1, where the antigen isa recombinant antigen.
 16. The method of claim 1, where the antigen is achimeric antigen.
 17. The method of claim 1, wherein the first cell typeis a cell that naturally expresses the cell-surface antigen.
 18. Themethod of claim 1, wherein the first cell type is a recombinant cellthat is engineered to heterologously express the cell-surface antigen.19. The method of claim 1, wherein the first cell type is adisease-associated variant of a normal cell, and optionally wherein thefirst cell type is a tumor cell.
 20. (canceled)
 21. The method of claim1, wherein the binding polypeptide is an antibody of fragment thereof,and optionally wherein the binding polypeptide is an antibody VH or VLdomain.
 22. (canceled)